Rotavirus C: prevalence in suckling piglets and development of virus-like particles to assess the influence of maternal immunity on the disease development.

Rotavirus C (RVC) has been detected increasingly in humans and swine in different countries, including the US. It is associated with significant economic losses due to diarrheal disease in nursing piglets. In this study we aimed: (1) to determine the prevalence of RVC in healthy and diarrheic suckling piglets on US farms; and (2) to evaluate if maternal antibody (Ab) levels were associated with protection of newborn suckling piglets against RVC. There was a significantly higher prevalence (p = 0.0002) of litters with diarrhea born to gilts compared with those born to multiparous sows. Of 113 nursing piglet fecal samples tested, 76.1% were RVC RNA positive. Fecal RVC RNA was detected in significantly (p = 0.0419) higher quantities and more frequently in piglets with diarrhea compared with healthy ones (82.5 vs. 69.9%). With the exception of the historic strain Cowden (G1 genotype), field RVC strains do not replicate in cell culture, which is a major impediment for studying RVC pathogenesis and immunity. To circumvent this, we generated RVC virus-like particles (VLPs) for Cowden (G1), RV0104 (G3) and RV0143 (G6) and used them as antigens in ELISA to detect swine RVC Abs in serum and milk from the sows. Using RVC-VLP Ab ELISA we demonstrated that sows with diarrheic litters had significantly lower RVC IgA and IgG Ab titers in milk compared to those with healthy litters. Thus, our data suggest that insufficient lactogenic protection provided by gilts plays a key role in the development of and the increased prevalence of clinical RVC disease.

Cholinergic enzymes and inflammatory markers in rats infected by Sporothrix schenckii.

The aim of this study was to evaluate the cholinesterase activity in serum, whole blood, and lymphocytes, as well as to verify its relation to immune response in rats experimentally infected by Sporothrix schenckii. For this study, 63 Wistar rats (Rattus norvegicus), male, adult were divided into three groups: the negative control group (GC: n = 21), the group infected subcutaneously (GSC: n = 21), and the group infected intraperitoneally (GIP: n = 21). The groups were divided into subgroups and the following variables were evaluated at 15, 30, and 40 days post-infection (PI): acetylcholinesterase (AChE) activity in lymphocytes and whole blood, butyrylcholinesterase (BChE) activity in serum, cytokines levels (IL-1, IL-6, TNFα, and INF-γ), immunoglobulins levels (IgA, IgG, IgM, and IgE), and protein profile by electrophoresis. Both infected groups showed increased levels of inflammatory parameters (P < 0.05) in tissue and inflammatory infiltrates. The activities of AChE in lymphocytes and BChE in serum increased (P < 0.05) significantly in animals from the GSC group on day 40 PI compared to the GC group. Regarding the GIP, there was a marked increase in the AChE activity in lymphocytes on days 30 and 40 PI, and in whole blood on days 15, 30, and 40 PI compared to GC. Furthermore, IL-10, an anti-inflammatory cytokine, was also present in high levels during chronic systemic S. schenckii infections in animals. Therefore, it is concluded that cholinesterase has an important modulatory role in the immune response during granulomatous infection by S. schenckii.

NTPDase and 5'-nucleotidase activities in synaptosomes of rabbits experimentally infected with BoHV-5.

Bovine herpesvirus type 5 (BoHV-5) is the causative agent of herpetic meningoencephalitis in cattle. The purinergic system is described as a modulator of the immune response and neuroinflammation. These functions are related to the extracellular nucleotides concentration. NTPDase and 5'-nucleotidase are enzymes responsible for controlling the extracellular concentration of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (ADO). The aim of this study is to determinate the ectonucleotidase activity in cortical synaptosomes and synaptosomes from the hippocampus of rabbits experimentally infected with BoHV-5. Rabbits were divided into four groups, two control groups (non-inoculated animals), and two infected groups (inoculated with BoHV-5). The infected groups received 0.5 ml of BoHV-5 suspension with 10(7.5)TCID50 of viral strain SV-507/99, per paranasal sinuses, and the control groups received 0.5 ml of minimum essential media per paranasal sinuses. Animals were submitted to euthanasia on days 7 and 12 post-inoculation (p.i.); cerebral cortex and hippocampus were collected for the synaptosomes isolation and posterior determination of the ectonucleotidase activities. The results showed a decrease (P < 0.05) in ectonucleotidase activity in synaptosomes from the cerebral cortex of infected rabbits, whereas an increased (P < 0.05) ectonucleotidase activity was observed in synaptosomes from the hippocampus. These differences may be related with the heterogeneous distribution of ectonucleotidases in the different brain regions and also with the viral infectivity. Therefore, it is possible to speculate that BoHV-5 replication results in changes in ectonucleotidase activity in the brain, which may contribute to the neurological signs commonly observed in this disease.

Effect of tepoxalin on renal function and hepatic enzymes in dogs exposed to hypotension with isoflurane.

OBJECTIVE: To evaluate the possible renal and hepatic toxicity of tepoxalin in dogs exposed to hypotension during isoflurane anesthesia. STUDY DESIGN: Prospective, randomized experimental study. ANIMALS: Twenty adult mixed-breed dogs, weighing 18.8 ± 2.8 kg. METHODS: The animals received 10 mg kg(-1) tepoxalin orally 2 hours before the anesthetic procedure (PRE; n = 6), or 30 minutes after anesthesia (POST; n = 6), along with a control group (CON; n = 8), which were only anesthetized. The PRE and POST groups also received the same dose of tepoxalin for 5 days post-procedure. All dogs were anesthetized with propofol and maintained with isoflurane and the end-tidal isoflurane (Fe'Iso) was increased until mean arterial pressure decreased to 50-60 mmHg. These pressures were maintained for 60 minutes. Heart rate, arterial pressures and Fe'Iso were recorded at 0, 10 and every 10 minutes up to 60 minutes of hypotension. Blood gases, pH, electrolytes and bleeding time were analyzed before and at 30 and 60 minutes of hypotension. Renal and hepatic changes were quantified by serum and urinary biochemistry and creatinine clearance. RESULTS: Serum concentrations of alanine amino transferase (ALT), alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT), blood urea nitrogen (BUN) and creatinine (Cr), and urinary output, urinary Cr, Cr clearance, and GGT:Cr ratio remained stable throughout the evaluations. During the anesthetic procedure there were no important variations in the physiological parameters. No side effects were observed in any of the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Tepoxalin did not cause significant effects on renal function or cause hepatic injury in healthy dogs exposed to hypotension with isoflurane, when administered pre- or postanesthetic and continued for five consecutive days.

Maternal immunization and vitamin A sufficiency impact sow primary adaptive immunity and passive protection to nursing piglets against porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea virus (PEDV) causes a highly contagious enteric disease with major economic losses to swine production worldwide. Due to the immaturity of the neonatal piglet immune system and given the high virulence of PEDV, improving passive lactogenic immunity is the best approach to protect suckling piglets against the lethal infection. We tested whether oral vitamin A (VA) supplementation and PEDV exposure of gestating and lactating VA-deficient (VAD) sows would enhance their primary immune responses and boost passive lactogenic protection against the PEDV challenge of their piglets. We demonstrated that PEDV inoculation of pregnant VAD sows in the third trimester provided higher levels of lactogenic protection of piglets as demonstrated by >87% survival rates of their litters compared with <10% in mock litters and that VA supplementation to VAD sows further improved the piglets' survival rates to >98%. We observed significantly elevated PEDV IgA and IgG antibody (Ab) titers and Ab-secreting cells (ASCs) in VA-sufficient (VAS)+PEDV and VAD+VA+PEDV sows, with the latter maintaining higher Ab titers in blood prior to parturition and in blood and milk throughout lactation. The litters of VAD+VA+PEDV sows also had the highest serum PEDV-neutralizing Ab titers at piglet post-challenge days (PCD) 0 and 7, coinciding with higher PEDV IgA ASCs and Ab titers in the blood and milk of their sows, suggesting an immunomodulatory role of VA in sows. Thus, sows that delivered sufficient lactogenic immunity to their piglets provided the highest passive protection against the PEDV challenge. Maternal immunization during pregnancy (± VA) and VA sufficiency enhanced the sow primary immune responses, expression of gut-mammary gland trafficking molecules, and passive protection of their offspring. Our findings are relevant to understanding the role of VA in the Ab responses to oral attenuated vaccines that are critical for successful maternal vaccination programs against enteric infections in infants and young animals.

Trypanosoma evansi infection impairs memory, increases anxiety behaviour and alters neurochemical parameters in rats.

The aim of this study was to investigate neurochemical and enzymatic changes in rats infected with Trypanosoma evansi, and their interference in the cognitive parameters. Behavioural assessment (assessment of cognitive performance), evaluation of cerebral L-[3H]glutamate uptake, acetylcholinesterase (AChE) activity and Ca+2 and Na+, K+-ATPase activity were evaluated at 5 and 30 days post infection (dpi). This study demonstrates a cognitive impairment in rats infected with T. evansi. At 5 dpi memory deficit was demonstrated by an inhibitory avoidance test. With the chronicity of the disease (30 dpi) animals showed anxiety symptoms. It is possible the inhibition of cerebral Na+, K+-ATPase activity, AChE and synaptosomal glutamate uptake are involved in cognitive impairment in infected rats by T. evansi. The understanding of cerebral host­parasite relationship may shed some light on the cryptic symptoms of animals and possibly human infection where patients often present with other central nervous system (CNS) disorders.

Increased cytokine and nitric oxide levels in serum of dogs experimentally infected with Rangelia vitalii.

This study aimed to measure the levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), and nitrite/nitrate (NO x ) in serum of dogs experimentally infected with Rangelia vitalii. Twelve female mongrel dogs were divided into 2 groups; group A (uninfected controls) composed by healthy dogs (n=5) and group B consisting of dogs inoculated with R. vitalii (n=7). Animals were monitored by blood smear examinations, which showed intraerythrocytic forms of the parasite on day 5 post-infection (PI). Blood samples were collected through the jugular vein on days 0, 10, and 20 PI to determine the serum levels of IFN-γ, TNF-α, IL-1, IL-6, and NO x . Cytokines were assessed by ELISA quantitative sandwich technique, and NO x was measured by the modified Griess method. Cytokine levels (IFN-γ, TNF-α, IL-1, and IL-6) were increased (P<0.01) in serum of infected animals. Serum levels of NO x were also increased on days 10 PI (P<0.01) and 20 PI (P<0.05) in infected animals. Therefore, the infection with R. vitalii causes an increase in proinflammatory cytokines and nitric oxide content. These alterations may be associated with host immune protection against the parasite.

Nitric oxide level, protein oxidation and antioxidant enzymes in rats infected by Trypanosoma evansi.

The aim of this study was to evaluate the nitric oxide (NO()) level, protein oxidation and antioxidant enzymes in rats infected with Trypanosoma evansi and establish the association of NO() levels with the degree of parasitemia. Thirty-six male rats (Wistar) were divided into two groups with 18 animals each. Group A was not infected while Group B was intraperitoneally infected, receiving 7.5×10(6) trypomastigotes per animal. Each group was divided into three subgroups with 6 rats each and blood was collected during different periods post-infection (PI), as follows: day 5 (A(5) and B(5)), day 15 (A(15) and B(15)) and day 30 PI (A(30) and B(30)). Blood samples were collected by cardiac puncture to estimate the levels of nitrites/nitrates (NO(x)) and advanced oxidation protein products (AOPP) in serum, and superoxide dismutase (SOD) and catalase (CAT) activities in blood. On days 15 and 30 PI NO(x) and AOPP levels were increased in serum of rats infected. Rodents infected with T. evansi showed a significant increase in SOD (days 5 and 15 PI) and CAT (day 30 PI) activities. Based on the physiological role of NO(), we can conclude that its increased concentration is related to an inflammatory response against the parasite, once a redox imbalance was observed during infection.

Activities of ectonucleotidases and adenosine deaminase in platelets of dogs experimentally infected with Rangelia vitalii.

Rangeliosis is a disease which affects dogs in Brazil, caused by a piroplasm known as Rangelia vitalii. This disease causes a lot of clinico-pathological features, including the coagulation disorders associated with bleeding. The cause of these changes has not yet been determined. Considering the association of purinergic system and hemostasis this study aimed to evaluate the activity of enzymes that hydrolyze ATP, ADP and AMP; and deamination of adenosine in platelets from dogs experimentally infected with R. vitalii. For this study, 12 healthy young dogs (females) were used, separated in two groups. Group A (n=5) were uninfected controls, and group B were experimentally infected with R. vitalii (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite after five days post-inoculation (PI). Blood samples were collected to quantitate and separate platelets (Day 0, 12 and 21 PI) and to measure the enzymatic activities (Day 12 and 21 PI). The activity of NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) was measured in platelets. A reduction (P<0.01) in the number of platelets was observed in R. vitalii-infected blood at Days 12 and 21 PI. At Day 12 PI, a reduction (P<0.01) in the hydrolysis of ATP, ADP and AMP, and deamination of adenosine was observed in dogs infected with R. vitalii. At Day 21 PI the ADA activity remained decreased, unlike the activity of NTPDase which increased (P<0.05). Based on these results we can conclude that ATP, ADP and AMP hydrolysis and adenosine deamination were altered in platelets of R. vitalii-infected dogs. Considering the importance of the purinergic system in hemostasis, it is believed that those changes contribute to the coagulation disorders and bleeding observed in R. vitalii-infected dogs and discussed in this manuscript.

Biochemistry detection of acetylcholinesterase activity in Trypanosoma evansi and possible functional correlations.

Several chemical and immunohistochemical techniques can be used for the detection of acetylcholinesterase (AChE) activity. In this experiment we aimed to detect AChE activity in Trypanosoma evansi. For this, the parasites were isolated from the blood of experimentally infected rats using a DEA-cellulose column. Enzymatic activity was determined in trypomastigote forms at 0, 0.2, 0.4, 0.8 and 1.2 mg/mL of protein concentrations by a standard biochemical protocol. At all concentrations tested, the study showed that T. evansi expresses the enzyme AChE and its activity was proportional to the concentration of protein, ranging between 0.64 and 2.70 µmol of AcSCh/h. Therefore, we concluded that it is possible to biochemically detect AChE in T. evansi, an enzyme that may be associated with vital functions of the parasite and also can be related to chemotherapy treatments, as further discussed in this article.

Cholinesterase as inflammatory markers in a experimental infection by Trypanosoma evansi in rabbits.

The aim of this study is to evaluate the role of cholinesterases as an inflammatory marker in acute and chronic infection by Trypanosoma evansi in rabbits experimentally infected. Twelve adult female New Zealand rabbits were used and divided into two groups with 6 animals each: control group (rabbits 1-6) and infected group (rabbits 7-12). Infected group received intraperitoneally 0.5 mL of blood from a rat containing 108 parasites per animal. Blood samples used for cholinesterases evaluation were collected on days 0, 2, 7, 12, 27, 42, 57, 87, 102 and 118 days post-inoculation (PI). Increased activity (P<0.05) of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) were observed in the blood on days 7 and 27, respectively and no differences were observed in cholinesterase activity in other periods. No significant difference in AChE activity (P>0.05) was observed in the encephalic structures. The increased activities of AChE and BChE probably have a pro-inflammatory purpose, attempting to reduce the concentration of acetylcholine, a neurotransmitter which has an anti-inflammatory property. Therefore, cholinesterase may be inflammatory markers in infection with T. evansi in rabbits.

Escherichia coli Nissle 1917 Enhances Efficacy of Oral Attenuated Human Rotavirus Vaccine in a Gnotobiotic Piglet Model.

Human rotavirus (HRV) infection is a major cause of viral gastroenteritis in young children worldwide. Current oral vaccines perform poorly in developing countries where efficacious vaccines are needed the most. Therefore, an alternative affordable strategy to enhance efficacy of the current RV vaccines is necessary. This study evaluated the effects of colonization of neonatal gnotobiotic (Gn) pigs with Escherichia coli Nissle (EcN) 1917 and Lacticaseibacillus rhamnosus GG (LGG) probiotics on immunogenicity and protective efficacy of oral attenuated (Att) HRV vaccine. EcN-colonized pigs had reduced virulent HRV (VirHRV) shedding and decreased diarrhea severity compared with the LGG-colonized group. They also had enhanced HRV-specific IgA antibody titers in serum and antibody secreting cell numbers in tissues pre/post VirHRV challenge, HRV-specific IgA antibody titers in intestinal contents, and B-cell subpopulations in tissues post VirHRV challenge. EcN colonization also enhanced T-cell immune response, promoted dendritic cells and NK cell function, reduced production of proinflammatory cytokines/Toll like receptor (TLR), and increased production of immunoregulatory cytokines/TLR expression in various tissues pre/post VirHRV challenge. Thus, EcN probiotic adjuvant with AttHRV vaccine enhances the immunogenicity and protective efficacy of AttHRV to a greater extent than LGG and it can be used as a safe and economical oral vaccine adjuvant.

Trypanosoma evansi: concentration of 3-nitrotyrosine in the brain of infected rats.

Nitric oxide (NO) is involved in many physiological processes, such as blood pressure control, neurotransmission, inhibition of platelet and neutrophil adherence, and the ability to kill tumor cells and parasites. The indirect determination of NO can be made by detection of 3-nitrotyrosine (3-NT) residues. The aim of this study was to measure the concentration of 3-NT in the brain of rats experimentally infected with Trypanosoma evansi. Twenty-four were inoculated intraperitoneally with cryopreserved blood containing 1×10(6) trypomastigotes per animal. Twenty-four animals were used as negative controls and received 0.2 mL of saline by the same route. The experimental groups (group C and T) were established according to the time after infection and the degree of parasitemia as follows: four control subgroups (C3, C5, C10 and C20) with six non-inoculated animals each and four test subgroups (T3, T5, T10 and T20) with six animals infected with T. evansi in each group. The animals were anesthetized with isoflurane and subsequently euthanized at the days 3 (C3, T3), 5 (C5, T5), 10 (C10, T10) and 20 (C20, T20) post-infection (PI). The brain was removed and dissected into cerebellum, cerebral cortex, striatum and hippocampus. Concentration of 3-NT in the brain was determined by Slot blot technique. At the day 3 PI no changes were observed in the concentration of 3-NT among the groups. There was a significant reduction (p<0.05) of 3-NT concentration in the striatum and cerebellum at the days 5 and 10 PI, respectively. At the day 20 PI a significant increase (p<0.05) of 3-NT was observed in the cerebellum, cerebral cortex and hippocampus from the infected animals. Therefore, T. evansi infection caused changes in the concentrations of 3-NT in the central nervous system (CNS), which may be related to clinical signs and infection management.

Cytokines in rats experimentally infected with Trypanosoma evansi.

The aim of this study was to measure the levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1) and interleukin 6 (IL-6) in the serum of rats experimentally infected with Trypanosoma evansi and to correlate these levels with hematological parameters. Initially, 48 rats (group T) were intraperitoneally inoculated with cryopreserved blood containing 1×10(6) trypomastigotes per animal. Twenty-eight animals (group C) were used as negative controls and received 0.2 mL of saline by the same route. The experimental groups were formed according to the time after infection and the degree of parasitemia as follows: four control subgroups (C3, C5, C10 and C20) with seven non-inoculated animals each and four test subgroups (T3, T5, T10 and T20) with 10 animals each inoculated with T. evansi. The blood samples were collected by cardiac puncture at days 3 (C3, T3), 5 (C5, T5), 10 (C10, T10) and 20 (C20, T20) post-infection (PI) to perform the complete blood count and the determination of IFN-γ, TNF-α, IL-1 and IL-6 levels using an ELISA quantitative sandwich. Infected rats showed normocytic normochromic anemia during the experimental period. T. evansi infection in rats caused a serum increase (P<0.01) of IFN-γ, TNF-α, IL-1 and IL-6 levels at days 3, 5, 10 and 20 PI compared to the controls. The multiple linear regressions showed a reduction of 24% in the hematocrit as a consequence of the increased IFN-γ, TNF-α and IL-1. Therefore, we conclude that the infection caused by T. evansi causes an increase in the pro-inflammatory cytokines. These results suggest a synergism among IL-1, TNF-α and IFN-γ contributing to the development of anemia. This increase is associated with the regulation of immune responses against the parasite.

Experimental infection with Rangelia vitalii in dogs: acute phase, parasitemia, biological cycle, clinical-pathological aspects and treatment.

Recently we conducted the molecular characterization of Rangelia vitalii, a protozoan with high pathogenicity for young dogs in southern Brazil. To date, the descriptions of the disease have been restricted to natural infection cases. Therefore, this study aimed to evaluate the parasitemia, biological cycles and clinical-pathological findings in dogs experimentally infected with R. vitalii in the acute phase of disease, and also aimed to test a therapeutic protocol based on the diminazene aceturate. For this study, we used 12 young dogs (females), separated into two groups. Group A was composed of healthy dogs, not-infected (n=5), and Group B consisted of animals infected with R. vitalii (n=7). After infection, the animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite 5 days post-infection (PI). Parasitemia increased progressively in these animals and had the highest peak of circulating parasites between 9 and 11 days PI. Subsequently, the parasitemia reduced and the protozoan was seen inside the leukocytes in days 17, 19 and 21 PI. The most prominent clinical signs observed at the 20 day PI of experiment were lethargy, fever and anorexia. We observed a decrease of hematocrit of infected animals compared with not-infected dogs, featuring a moderate anemia. Pathological evaluation of one dog in Group B at day 21 PI revealed splenomegaly, hepatomegaly, lymphadenopathy, and hemorrhages at necropsy. Histological examination showed only follicular hyperplasia in the spleen and lymph nodes, and the etiologic agent in the vascular endothelium. At 21 days PI, it was performed the treatment of dogs in Group B (n=6) with a single dose of diminazene aceturate, which showed a curative efficacy of 100% in cleaning R. vitalii from blood of infected dogs.

Escherichia coli Nissle 1917 Enhances Innate and Adaptive Immune Responses in a Ciprofloxacin-Treated Defined-Microbiota Piglet Model of Human Rotavirus Infection.

Human rotavirus (HRV) infection is a major cause of gastroenteritis in children worldwide. Broad-spectrum antibiotic-induced intestinal microbial imbalance and the ensuing immune-metabolic dysregulation contribute to the persistence of HRV diarrhea. Escherichia coli Nissle 1917 (EcN), a Gram-negative probiotic, was shown to be a potent immunostimulant and alleviated HRV-induced diarrhea in monocolonized gnotobiotic (Gn) piglets. Our goal was to determine how EcN modulates immune responses in ciprofloxacin (Cipro)-treated Gn piglets colonized with a defined commensal microbiota (DM) and challenged with virulent HRV (VirHRV). Cipro given in therapeutic doses for a short term reduced serum and intestinal total and HRV-specific antibody titers, while EcN treatment alleviated this effect. Similarly, EcN treatment increased the numbers of total immunoglobulin-secreting cells, HRV-specific antibody-secreting cells, activated antibody-forming cells, resting/memory antibody-forming B cells, and naive antibody-forming B cells in systemic and/or intestinal tissues. Decreased levels of proinflammatory but increased levels of immunoregulatory cytokines and increased frequencies of Toll-like receptor-expressing cells were evident in the EcN-treated VirHRV-challenged group. Moreover, EcN treatment increased the frequencies of T helper and T cytotoxic cells in systemic and/or intestinal tissues pre-VirHRV challenge and the frequencies of T helper cells, T cytotoxic cells, effector T cells, and T regulatory cells in systemic and/or intestinal tissues postchallenge. Moreover, EcN treatment increased the frequencies of systemic and mucosal conventional and plasmacytoid dendritic cells, respectively, and the frequencies of systemic natural killer cells. Our findings demonstrated that Cipro use altered immune responses of DM-colonized neonatal Gn pigs, while EcN supplementation rescued these immune parameters partially or completely.IMPORTANCE Rotavirus (RV) is a primary cause of malabsorptive diarrhea in children and is associated with significant morbidity and mortality, especially in developing countries. The use of antibiotics exacerbates intestinal microbial imbalance and results in the persistence of RV-induced diarrhea. Probiotics are now being used to treat enteric infections and ulcerative colitis. We showed previously that probiotics partially protected gnotobiotic (Gn) piglets against human RV (HRV) infection and decreased the severity of diarrhea by modulating immune responses. However, the interactions between antibiotic and probiotic treatments and HRV infection in the context of an established gut microbiota are poorly understood. In this study, we developed a Gn pig model to study antibiotic-probiotic-HRV interactions in the context of a defined commensal microbiota (DM) that mimics aspects of the infant gut microbiota. Our results provide valuable information that will contribute to the treatment of antibiotic- and/or HRV-induced diarrhea and may be applicable to other enteric infections in children.

Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota.

Human rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in low- and middle-income countries (LMICs), where HRV vaccine efficacy is low. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of enteric diseases in humans. However, repeated doses of EcN are required to achieve maximum beneficial effects. Administration of EcN on a microsphere biofilm could increase probiotic stability and persistence, thus maximizing health benefits without repeated administrations. Our aim was to investigate immune enhancement by the probiotic EcN adhered to a dextranomar microsphere biofilm (EcN biofilm) in a neonatal, malnourished piglet model transplanted with human infant fecal microbiota (HIFM) and infected with rotavirus. To create malnourishment, pigs were fed a reduced amount of bovine milk. Decreased HRV fecal shedding and protection from diarrhea were evident in the EcN biofilm treated piglets compared with EcN suspension and control groups. Moreover, EcN biofilm treatment enhanced natural killer cell activity in blood mononuclear cells (MNCs). Increased frequencies of activated plasmacytoid dendritic cells (pDC) in systemic and intestinal tissues and activated conventional dendritic cells (cDC) in blood and duodenum were also observed in EcN biofilm as compared with EcN suspension treated pigs. Furthermore, EcN biofilm treated pigs had increased frequencies of systemic activated and resting/memory antibody forming B cells and IgA+ B cells in the systemic tissues. Similarly, the mean numbers of systemic and intestinal HRV-specific IgA antibody secreting cells (ASCs), as well as HRV-specific IgA antibody titers in serum and small intestinal contents, were increased in the EcN biofilm treated group. In summary EcN biofilm enhanced innate and B cell immune responses after HRV infection and ameliorated diarrhea following HRV challenge in a malnourished, HIFM pig model.

Comparative Sequence Analysis of Historic and Current Porcine Rotavirus C Strains and Their Pathogenesis in 3-Day-Old and 3-Week-Old Piglets.

The increased prevalence of porcine group C rotavirus (PRVC) in suckling piglets and the emergence of new genetically distinct PRVC strains are concerning due to the associated significant economic losses they cause to the swine industry. We sequenced and analyzed two new PRVC strains, RV0104 (G3), and RV0143 (G6) and compared their pathogenesis with that of the historic strain Cowden (G1) in gnotobiotic (Gn) pigs. Near complete genome sequence analysis confirmed that these two strains were distinct from one another and the Cowden strain. VP1, VP2, VP6, NSP1-NSP3, and NSP5 genes were more similar between Cowden and RV0143, whereas VP3, VP7, and NSP4 shared higher nucleotide identity between Cowden and RV0104. Three-day-old and 3-week-old Gn piglets were inoculated with 105 FFU/piglet of Cowden, RV0104 or RV0143, or mock. All 3-day-old piglets developed severe diarrhea, anorexia, and lethargy, with mean PRVC fecal shedding titers peaking and numerically higher in RV0104 and RV0143 piglets on post infection day (PID) 2. Histopathological examination of the small intestine revealed that the 3-day-old Cowden and RV0104 inoculated piglets were mildly affected, while significant destruction of small intestinal villi was observed in the RV0143 inoculated piglets. Consistent with the highest degree of pathological changes in the small intestines, the RV0143 inoculated piglets had numerically higher levels of serum IL-17 and IFN-α cytokines and numerically lower PRVC IgA geometric mean antibody titers. Milder pathological changes and overall higher titers of PRVC IgA antibodies were observed in 3-week-old vs. 3-day-old piglets. Additionally, diarrhea was only observed in RV0104 and RV0143 (but not Cowden) inoculated 3-week-old piglets, while levels of serum IL-10 and PRVC IgA antibodies were higher in Cowden inoculated pigs, consistent with the lack of diarrhea. Thus, we confirmed that these current, genetically heterogeneous PRVC strains possess distinct pathobiological characteristics that may contribute to the increased prevalence of PRVC diarrhea in neonatal suckling piglets.

Malnutrition Decreases Antibody Secreting Cell Numbers Induced by an Oral Attenuated Human Rotavirus Vaccine in a Human Infant Fecal Microbiota Transplanted Gnotobiotic Pig Model.

Human rotavirus (HRV) is a leading cause of morbidity and mortality in children, especially in developing countries. Malnutrition is prevalent in these countries, which may contribute to the decreased oral vaccine efficacy, posing a concern for global health. Neonatal gnotobiotic (Gn) pigs closely resemble human infants in their anatomy, physiology, and outbred status and are a unique model to investigate malnutrition, oral live attenuated HRV (AttHRV) vaccination, and subsequent virulent HRV (VirHRV) challenge. We evaluated the impact of malnutrition on AttHRV vaccine efficacy and B cell immune responses in neonatal germfree (GF) or Gn pigs transplanted with human infant fecal microbiota (HIFM). Pigs were fed either deficient or sufficient bovine milk diets. Malnutrition did not significantly affect the serum and intestinal contents total or HRV-specific IgG and IgA antibody titers pre VirHRV challenge. However, HRV-specific IgG and IgA antibody secreting cells (ASCs) were reduced in blood or intestinal tissues following AttHRV vaccination and pre VirHRV challenge in deficient HIFM transplanted pigs. Furthermore, post-VirHRV challenge, deficient HIFM pigs had decreased total Ig and HRV-specific IgG and IgA antibody titers in serum or intestinal contents, in addition to decreased HRV-specific IgG and IgA ASCs in blood and ileum, compared with sufficient HIFM pigs. Our results indicate that deficient diet impairs B cell mucosal, and systemic immune responses following HRV vaccination, and challenge. The impaired immune responses contributed to the decreased protective efficacy of the AttHRV vaccine, suggesting that malnutrition may significantly reduce the effectiveness of oral HRV vaccines in children in developing countries.

Epidemiology of Deltacoronaviruses (δ-CoV) and Gammacoronaviruses (γ-CoV) in Wild Birds in the United States.

Porcine deltacoronavirus (δ-CoV) is the object of extensive research in several countries including the United States. In contrast, the epidemiology of δ-CoVs in wild birds in the US is largely unknown. Our aim was to comparatively assess the prevalence of δ- and γ-CoVs in wild migratory terrestrial and aquatic birds in Arkansas, Illinois, Indiana, Maryland, Mississippi, Missouri, Ohio, Tennessee and Wisconsin. A total of 1236 cloacal/fecal swabs collected during the period 2015-2018 were tested for γ- and δ-CoVs using genus-specific reverse transcription-PCR assays. A total of 61 (4.99%) samples were γ-CoV positive, with up to 29 positive samples per state. In contrast, only 14 samples were positive for δ-CoV (1.14%) with only 1-4 originating from the same state. Thus, unlike previous reports from Asia, γ-CoVs are more prevalent than δ-CoVs in the US, suggesting that δ-CoVs may spread in birds with lower efficiency. This may indicate δ-CoV emerging status and incomplete adaptation to new host species limiting its spread. Phylogenetic analysis of the partial N gene revealed that the newly identified δ-CoV strains were most closely related to the HKU20 (wigeon) strain. Further studies are necessary to investigate the role of aquatic bird δ-CoVs in the epidemiology of δ-CoVs in swine and terrestrial birds.